Antimicrobial and Antibiofilm Activity of Monolaurin against Methicillin-Resistant Staphylococcus aureus Isolated from Wound Infections

Background Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major pathogens associated with life-threatening infections, showing resistance to various antibiotics. This study aimed to assess the influence of monolaurin on biofilm-forming MRSA. Methods The agar dilution method determined the minimum inhibitory concentration (MIC) of monolaurin against MRSA isolates and explored its impact on the resistance profile of selected antibiotics. The assessment of combined therapy involving monolaurin and antibiotics was conducted using fractional inhibitory concentration (FIC). The tissue culture plate strategy appraised monolaurin's antibiofilm activity and its inhibitory concentration (IC50), with assessment via scanning electron microscopy. Reverse transcription polymerase chain reaction (RT-PCR) discerned a monolaurin effect on the expression of the icaD gene. Results Monolaurin exhibited MIC values ranging from 500 to 2000 μg/mL. FIC index showed a synergistic effect of monolaurin with β-lactam antibiotics ranging from 0.0039 to 0.25 (p < 0.001). Among the 103 investigated MRSA isolates, 44 (44.7%) displayed moderate biofilm formation, while 59 (55.3%) were strong biofilm producers. Antibiofilm activity demonstrated concentration dependence, confirming monolaurin's capacity to inhibit biofilm formation and exhibited strong eradicating effects against preformed MRSA biofilms with IC50 values of 203.6 μg/mL and 379.3 μg/mL, respectively. Scanning electron microscope analysis revealed reduced cell attachments and diminished biofilm formation compared to the control. The expression levels of the icaD gene were remarkably reduced at monolaurin concentrations of 250 and 500 μg/mL. Conclusion Monolaurin had significant inhibitory effects on MRSA pre-existing biofilms as well as biofilm development. So, it can be employed in the treatment of severe infections, particularly those associated with biofilm formation including catheter-associated infections.


Introduction
Te emergence of infections associated with multidrugresistant (MDR) microorganisms is a serious global issue and a great challenge for healthcare teams worldwide [1].
Over recent years, there has been a notable increase in the incidence of Staphylococcus aureus infections, especially those infections caused by MDR Staphylococci that exhibit heightened morbidity and mortality compared to infections caused by other bacteria [2].Infections with S. aureus can range from minor dermatological infections to major illnesses such as septicemia, toxin-mediated toxic shock syndrome, endocarditis, pneumonia, and osteomyelitis.Besides, it can cause chronic skin infections and mastitis in farm animals [3,4].Te threat posed by S. aureus infections is increased by the emergence of methicillin-resistant S. aureus (MRSA) and nonspecifc resistance mechanisms including bioflms.Te development of bioflms on either host tissues, such as implanted materials, including dialysis catheters, orthopedic implants, prosthetic heart valves, urine catheters, and central venous catheters, has been implicated in many of these pathological conditions [5,6].A considerable proportion of infections, those associated with embedded medical devices, was thought to be caused by microbial bioflms, as indicated by various estimations [7].Microbial bioflms are ubiquitous as the predominant life forms for microbes across various systems [8].Bacterial bioflms are complex communities encased within a selfproduced extracellular matrix primarily composed of polysaccharides, proteins, lipids, and extracellular DNA (eDNA).Tis matrix plays a crucial role in protecting bioflm bacteria from host immune responses.Bioflm bacteria display emergent properties distinct from freeliving cells, including enhanced genetic exchange, formation of synergistic microconsortia, and increased resistance to antimicrobials, attributed largely to the EPS matrix [9,10].In addition, bioflms facilitate the spread of antibiotic resistance through horizontal gene transfer.However, detecting bacterial bioflms presents challenges due to their inherent tolerance to host defenses and conventional antibiotic treatments, complicating routine diagnostic procedures [11].Te treatment choices were also limited as a result of these bioflm's high resistance to host defenses and antimicrobial treatments [6].Te therapeutic management of MRSA in Egypt had encountered a considerable difculty with a concerning increase in prevalence over time necessitating efcient microbial control strategies that disrupt the antibiotic resistance cycle [12].Tus, the imperative to fnd bioflm inhibitors arises as a critical need from this circumstance.
Monolaurin is a byproduct of coconut oil, prepared from lauric acid and glycine.So, glycerol monolaurate (GML) is a fatty acid monoester.Te Food and Drug Administration (FDA) recognizes glycerol monolaurate (GML) as a safe, natural substance that is frequently used as an additive in cosmetic and food manufacturing.In addition, GML exhibits broad-spectrum antibacterial properties, particularly against Gram-positive bacteria including Gram-positive cocci.Qanash et al. [13] reported the antimicrobial efcacy of coconut oil nanoemulsion (NE) against some microorganisms including S. aureus and their ability to decrease infammation.Also, Khan et al. [14] elucidated GML's potential in reducing bioflm formation and as an antiquorum sensing agent.Monolaurin probably uses several diferent processes to work, though.Also, GML inhibits the generation of Gram-positive exotoxins and eliminates pre-existing S. aureus bioflms [15].Te use of monolaurin, which precisely targets key stages involved in bioflm formation and has shown antimicrobial activity, would be a prudent approach for lowering the risk of infections caused by S. aureus bioflms.Terefore, our study was conducted to assess the antimicrobial resistance patterns of MRSA strains, evaluate the potential impact of monolaurin on the antimicrobial susceptibility of the isolated MRSA strains, and ascertain its infuence on bioflm formation and preformed bioflm produced by the tested isolates.

Study Area, Design, and Population.
A cross-sectional observational study was carried out from September 2021 to April 2022 at Minia University Hospital (Minia, Egypt).A total of 155 clinical specimens were obtained from patients who were admitted to Minia University hospitals in Egypt with wound infections.Being part of standard hospital laboratory protocols, the clinical samples were collected and labeled with the patient's name and the source.Te Ethics Committee of the Deraya University's faculty of pharmacy approved the study's use of human participants.

Inclusion and Exclusion
Criteria.Te inclusion criteria comprised patients meeting the following conditions: (a) according to the following wound etiologies: surgical wounds, accidental wounds, abscesses, ulcers, and burns with positive (+) bacterial culture results; (b) age between 1 and 60 years; and (c) provision of informed consent by the patients.Conversely, the exclusion criteria encompassed the following: (a) patients exhibiting severe dysfunction of vital organs such as the heart, brain, liver, kidneys, or other major organs or sufering from severe primary diseases; (b) patients with underlying medical conditions impacting wound healing, including hematologic disorders, immune system disorders, and connective tissue diseases; (c) patients concurrently participating in other clinical studies; and (d) patients demonstrating inadequate compliance.

Bacterial Investigation.
Samples were cultured on brain heart infusion broth at 37 °C for 24 hours.For isolation and purifcation, mannitol salt agar was streaked with a loopful of bacterial suspension and incubated for 24 hours at 37 °C.S. aureus isolates were initially recognized through the appearance, fermentation of mannitol sugar, morphology, gram staining, and biochemical characteristics of the colony (tests for catalase, coagulase (tube and slide), and DNase).

Efect of Monolaurin on Bioflm Formation
(1) Bioflm Detection using Tissue Culture Plate (TCP) Method.According to Manandhar et al. [21], bacterial culture was cultivated at 37 °C for 24 h in tryptic soy broth (TSB) supplied with 1% (w/v) glucose, followed by a 1 : 100 dilution using fresh medium.Next, the diluted culture (200 μL) was dispensed into individual wells of a 96-well microtiter plate.Negative controls included uninoculated tryptic soy broth which was incubated for 48 h for the plates at 37 °C.Ten, the contents of each well were taken out, and the wells were washed three times with 200 μL of sterile, distilled water.Adherent bioflms were frst fxed for 15 minutes with 95% ethanol and then stained for 5 minutes with 100 μL of 1% crystal violet.After eliminating unbound dye, the wells were rinsed with 200 μL of sterile distilled water and air-dried.To dissolve the formed bioflm, 100 μL of ethanol was applied.Finally, the optical densities (ODs) of stained adherent bioflms were quantifed at a wavelength of 570 nm using a micro-ELISA auto reader.Tree duplicates of the experiment were conducted.Te average OD values of the sterile medium were omitted from all test results once they had been calculated.ODs below 0.120 were considered nonbioflm producers, ODs between 0.120 and 0.240 were considered moderate bioflm producers, and ODs greater than 0.240 were considered strong bioflm producers [22,23].
(2) Measuring the Efectiveness of Diferent Monolaurin Concentrations in Eradicating Bioflms.Te efcacy of monolaurin in disrupting preformed bioflms was investigated.A suspension of the tested strains (100 μL) was inoculated into individual wells of a 96-well tissue culture plate.Uninoculated TSB was considered as a negative control.Incubation of plates was at 37 °C for 24 h.Wells were then washed with 200 μL of phosphate bufer saline (PBS).Ten, 100 μL of each tested concentration of monolaurin (250-2000 μg/mL) was added to each well.Normal saline was added to control wells (positive control and negative control).Plates were incubated at 37 °C for 24 h.Supernatants were cast of, and the wells were washed at least twice by 200 μL of PBS.Bioflm was measured using crystal violet assay.At 570 nm, the bioflm's OD was determined after a 24-hour aerobic incubation period at 37 °C.Te mean bioflm OD at the used concentrations of monolaurin was found to be equal to or less than the OD of the negative control.Te percentage of inhibition of preformed bioflm was calculated by using the following formula: Te activity of monolaurin on the preformed bioflm against all MRSA isolates was expressed as the 50% inhibitory concentration (IC 50 ).Te percentage of inhibition was plotted against monolaurin concentration using Prism software (GraphPad Prism 9 software) to determine the IC 50 value [24,25].Trials were conducted in triplicate [26].

International Journal of Microbiology (3) Measuring the Efectiveness of Diferent Monolaurin
Concentrations on Bioflm Formation.Monolaurin's capability to inhibit bioflm formation was assessed.Te bacterial suspension (100 μL) was distributed in tissue culture plate wells.One hundred microliters of varying concentrations of monolaurin (250, 500, 1000, and 2000 μg/mL) were added to the wells containing bacterial suspension, while bioflms were allowed to grow as previously mentioned.Plates were incubated at 37 °C for 24 h.Supernatants were discarded, and wells were washed twice by PBS.Te efect of monolaurin on bioflm formation was detected by crystal violet assay at 570 nm.As a positive control, MRSA isolate culture was used, while the negative control was uninoculated tryptic soy broth (Oxoid, USA).IC 50 value of monolaurin activity on bioflm formation was calculated as previously mentioned.Te experiments were performed three times [26].

Scanning Electron Microscope.
Following treatment with monolaurin, bioflm development was observed using a scanning electron microscope (SEM).Monolaurin and TSB were combined at 1×MIC and 2×MIC of the tested strains and then pipetted onto sterile culture plates containing MRSA.Untreated cells were used as control.Every dish had a sterile plastic coverslip in it.Following the instructions of the Huazhong Agricultural University's microscopy core laboratory, the coverslips were carefully cleaned twice with sterile PBS and then 2.5% glutaraldehyde/ PBS (v/v, pH 7.2) was applied.After that, the samples underwent ethanol dehydration.Te coverslips were carefully cleaned twice with sterile PBS following an overnight incubation at 37 °C.Using a Hitachi SU8010, MRSA isolates bioflm formation on the coverslips was detected [27].
2.8.Conventional PCR icaA and icaD Bioflm Genes.Te procedures were carried out in accordance with the manufacturer's instructions upon using a DNA extraction kit (QIAamp DNA Mini Kit, Germany).For the icaA gene, the oligonucleotide primer sequences (Metabion, Germany) were as follows: forward primer 5′ CCT AAC TAA CGA AAG GTA G 3′ and reverse primer 5′ AAG ATA TAG CGA TAA GTG C 3′ with amplicon size 1315 bp, and icaD gene, F 5′AAA CGT AAG AGA GGT GG 3′ and R 5′ GGC AAT ATG ATC AAG ATA3′ with amplicon size 381 bp [28].Te conditions used to conduct the polymerase chain reaction (PCR) are summarized in Supplementary Table (ST-1).Electrophoresis using 1.5% agarose gel for 30 minutes at a continuous current of 1-5 volts/cm was the applied technique for PCR product detection.DNA bands were identifed by ethidium bromide staining and UV transillumination light.

Real-Time Polymerase Chain Reaction (RT-PCR).
RT-PCR was performed to measure the relative expression of the icaD gene in the absence and presence of 0.25×MIC and 0.5×MIC of monolaurin (Supplementary Table ST-2 contains a list of the primer sequences).Each experimental and control tube was incubated at 37 °C for 24 h.Te RNeasy Mini Kit's instructions were carried out to extract total RNA.Te cycling conditions are shown in Supplementary Table (ST-3).Gene expression levels were standardized to 16S rRNA.Amplifcation curves and cycle threshold (CT) were computed using the start gene Mx3005P program.Te CT of each sample was compared with that of the control, and the variation in gene expression among RNA samples was assessed using the "Ct" approach proposed by Yuan et al. [29].Dissociation curves from several samples were evaluated to rule out false positive results.

Statistical
Analysis.SPSS version 25.0 software was utilized for data analysis.To determine changes in the ODs of bioflms with increasing monolaurin concentrations, the general linear model for repeated measurements (for not normally distributed data) was employed.Using Wilcoxon's test, treated bioflms with various concentrations of monolaurin were compared with untreated bioflms (control).

Staphylococcus aureus Isolation and Identifcation.
A total of 155 samples from patients of diferent ages, who had been admitted to the Minia University hospitals in Egypt with wound infection between September 2021 and April 2022 were examined for bacterial growth.Clinical samples included the following: 63 samples from surgical wounds, 41 samples from accidental wound infections, 20 samples from abscesses and ulcers, and 31 samples from burns.By using several culture and biochemical techniques, 115 (74.19%) of these collected samples were identifed as S. aureus bacterial isolates.

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International Journal of Microbiology  3). Figure 4 shows the efect of monolaurin on the inhibition of bioflm produced by the tested isolate.In comparison to control (untreated cells), it was found that treating cells with monolaurin resulted in fewer cell attachments and decreased bioflm development at concentrations of 1000 and 2000 μg/mL.

Conventional PCR.
Genomic analysis was conducted on 59 MRSA strains identifed as strong bioflm producers, isolated from diferent types of wound infections, with the aim of detecting the presence of the icaA and icaD genes.
Out of the tested samples, 52 (88.14%) samples were icaD positive, while all isolates were negative for the icaA gene as illustrated in Supplementary Figure (SF-4).Te distribution of icaD genes among diferent types of wound infection is illustrated in Supplementary Table (ST-7).

Efect of Monolaurin on Expression of icaD Gene among MRSA Strains.
Quantitative real-time PCR was employed to assess the gene expression of icaD in samples treated with monolaurin at 250 μg/mL and 500 μg/mL concentrations.Four isolates were chosen for testing the activity of monolaurin on the gene expression of the icaD gene.Upon using 250 μg/mL of monolaurin on the tested strains, a fold decrease in the expression of the icaD gene ranged from 25.26 to 38.44, while when using 500 μg/mL of monolaurin, fold change ranged from 62.11 to 87.76 compared to the positive control samples (Figure 5).

Discussion
Methicillin-resistant Staphylococcus aureus (MRSA) is a common antibiotic-resistant organism contributing signifcantly to both community and hospital-associated infections.Te fact that MRSA may develop bioflms on both International Journal of Microbiology biotic and abiotic surfaces adds complexity to the challenge posed by this organism.Staphylococci have been recognized for their ability to prompt bioflm-associated infections [30][31][32].Te resilience of established bioflms to various inhibitory techniques makes them particularly challenging to regulate or eradicate [33].Te development of efective antibioflm strategies is crucial not only for enhanced treatment of bioflm-related diseases but also for mitigating

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International Journal of Microbiology the acceleration and distribution of antibiotic resistance in afected individuals [34].Natural antibioflm compounds have been discovered as a of bioflm's increased antibiotic resistance rate [35].Monolaurin, a naturally occurring substance frequently present in coconut oil, exhibits strong antibacterial properties.Our investigation was   International Journal of Microbiology assumed to elucidate the potential impact of monolaurin on both bioflm development and preformed bioflms generated by the MRSA isolates.
In the present study, out of the 155 samples, 115 (74.19%) were positive for S. aureus.In 115 S. aureus samples, 103 (89.56%) were MRSA.Tis result was in alignment with the fndings of Rehman et al. [36], who reported that 78.3% of the clinical isolates of S. aureus collected from tertiary care hospitals in Peshawar, Pakistan, were MRSA.Alreeme et al. [37] highlighted Mycobacterium tuberculosis and Methicillin-resistant S. aureus as the most detectable microorganisms associated with respiratory diseases during Hajj.Our study revealed also a heightened incidence of MRSA (37.9%) in patients with accidental wound infection compared to other types of infections.Correspondingly, Garoy et al. [38] found MRSA detection rates of 62.5%, 60%, 78.6%, and 87.5% in patients with surgical wounds, abscesses, and burns, respectively.El Amin and Faidah [39] identifed MRSA as the most frequently isolated pathogen in wound infections.In addition, Alahmadi et al. [40] emphasized the rising issue of multidrug-resistant hospital-acquired MRSA in Saudi Arabia due to inappropriate antibiotic use.
Te antibiogram of the studied MRSA strains revealed that linezolid and imipenem were the most efective antibiotic against S. aureus (2% and 3% resistance rates, respectively) followed by vancomycin (4.35% resistance rate) and chloramphenicol (13.9% resistance rate).MRSA showed complete resistance to amoxicillin/clavulanic acid, piperacillin/tazobactam, ampicillin/sulbactam, and cefoxitin (100%) and moderate resistance to tetracycline (64.07%), rifampicin (40.77%), ciprofoxacin and levofoxacin (38.8%), and gentamicin (37.86%).Similar results were obtained by Rehman et al. [36], who indicated that MRSA isolates had 100% resistance to penicillin and cefoxitin and low resistance to both chloramphenicol (14.46%) and linezolid (2.41%), respectively.In another study performed by Jafari-Sales et al. [41], they showed that MRSA isolated from tertiary care hospitals in the North Batinah region, Oman, were completely resistant to penicillin and highly resistant to    International Journal of Microbiology Due to the high prevalence of MDR bacteria, numerous researchers tried alternative strategies to assess the impact of nonantimicrobial agents on the growth of these pathogens.Investigations have explored the potential efcacy of substances such as essential oils, plant extracts, and certain food supplements [45][46][47].Within the scope of our study, the MIC range for monolaurin against MRSA was determined to be 250-2000 μg/mL.Comparable results were observed in a study conducted by Nitbani et al. [48], in which monolaurin was found to inhibit the growth of S. aureus, including MRSA, even at the lowest concentration tested (500 μg/mL).Furthermore, when compared to standard strains S. aureus ATCC 25923 and ATCC 1885, monolaurin exhibited MICs of 100 and 250 μg/mL, respectively [49,50].
Drug combination therapy is a workable strategy, but it must be chosen carefully to prevent the emergence of resistance [51].Te FICi results for synergy confrmed the synergistic antibacterial activity of β-lactam and monolaurin antibiotics against MRSA isolates [52].It has been demonstrated that combining many antimicrobials can reduce the dosages needed for each antimicrobial while increasing their antibacterial activity [53].Te efective elimination of Staphylococcal infection in patients is greatly restricted by bioflms as they protect bacteria from the action of both the host immune system and antibacterial medications [54].Various methods exist for evaluating bacterial bioflm formation, with the microtiter plate bioflm formation assay acknowledged as a particularly efective technique.In our investigation, bioflm formation was assessed by the tissue culture plate (TCP) method, and the fndings revealed that 57.28% of MRSA isolates showed strong bioflms, while only 42.72% of the isolates produced moderate bioflms.Our fndings were in agreement with a research performed by Hosseini et al. [55], who reported that 52.9% and 45.3% of MRSA strains were strong and moderate bioflm producers, in respective order.Also, Awan et al. [56] found that all the tested strains of S. aureus produced bioflms showing 63.6% as strong bioflm producers and 36.4% as moderate bioflm producers.In contrast, Gaire et al. [57] reported divergent fndings, indicating that among 27 S. aureus strains, only 1 (4%) isolate exhibited strong bioflm production, 19 (70%) produced weak bioflms, and 7 (26%) produced moderate bioflms.Consequently, it appeared that the strains in our investigation exhibited heightened virulence and a greater propensity for strong bioflm production, potentially attributed to variations in sample sources or diferences in antibiotic administration practices in Egypt.
Te bioflms formed by the 103 MRSA strains involved in this study were examined using varying concentrations of monolaurin, specifcally at 250, 500, 1000, and 2000 μg/mL.Employing the tissue culture plate (TCP) method, crystal violet staining was utilized to quantify the efectiveness of monolaurin in inhibiting the formation of bioflms and eradicating those established by MRSA isolates.Te results demonstrated that monolaurin exhibited a dose-dependent inhibitory efect on MRSA bioflm formation and eradication.As depicted in Tables 2 and 3, an escalation in monolaurin concentration corresponded to an augmentation in the inhibitory activity against bioflm formation.Tese results concurred with those of Krislee et al. [47], who observed the efcient suppression of Staphylococcus epidermidis bioflms by monolaurin at concentrations ranging from 1 to 1.9 mg/mL.Furthermore, it has been reported that glycerol monolaurate, at a concentration of 1,822 μM, exhibited complete removal of S. aureus bioflms [58].Following Mochtar et al., the authors in [59] found that monolaurin can diminish S. epidermidis bioflm formation by 80% at 1,953 μg/mL.Also, monolaurin can suppress the development of bioflms by decreasing bacterial cell's hydrophobicity and preventing bacterial cell's adherence [60].
Using SEM analysis, we discerned the inhibitory and antibioflm efects of monolaurin on MRSA bioflm.In the control group, MRSA exhibited strong bioflm formation on the coverslip, characterized by aggregates and microcolonies.However, upon the introduction of 1000 and 2000 μg/mL concentrations of monolaurin into the culture conditions, a distinguished reduction in both bioflm cell count and extracellular matrix was observed.Our results were in accordance with a study performed by Gil et al. [61].Tey found that the absorbance values for coated wires with monolaurin were much lower than those for plain wires, indicating the antibioflm activity of monolaurin-coated wires against MSSA, MRSA, and S. epidermidis.Furthermore, SEM examination of S. epidermidis treated with monolaurin at concentrations ranging from 1000 to 1953 μg/ mL revealed diminished cell attachments and reduced bioflm production, supporting the inhibitory potential of monolaurin against bioflm formation [59].
Polysaccharide intercellular adhesin [62], which is made of β−1,6-N-acetylglucosamine and is responsible for cellto-cell adhesion, is expressed in S. aureus strains and regulates the formation of bioflms.Te chromosomal intercellular adhesion (ica) locus, comprising the icaADBC structural genes and the icaR regulatory gene, encodes PIA.Te icaA and icaD genes within this locus govern the synthesis of PIA, thereby regulating the bioflm-forming ability of S. aureus strains.Tis suggests the potential utility of the ica locus as a therapeutic target for bioflmassociated infections [63].Trough conventional PCR, we identifed the presence of both icaA and icaD genes in our study.Surprisingly, none of the examined isolates carried the icaA gene, while the icaD gene was detected in 88.14% of the tested MRSA isolates.Our results were consistent with observations of Darwish and Asfour [64], who noted that the icaA gene was less prevalent compared to the icaD gene, and the presence of either gene did not necessarily correlate with bioflm formation.
Real-time PCR analysis of the expression of icaD following treatment with 250 μg/mL and 500 μg/mL of monolaurin revealed a signifcant downregulation of gene expression following monolaurin treatment by 25.26-38.44and 62.11-87.76-foldchange, respectively, in comparison with their resultant levels prior to treatment.Consequently, monolaurin interfered with the expression of a gene associated with bioflm production (icaD).Our observations were found to be compatible with those of Schlievert and Peterson [58] and Nitbani et al. [65], whose research similarly indicated that monolaurin disrupts bioflm formation, 10 International Journal of Microbiology resulting in lower polysaccharide growth and reduced cell adhesion inside bioflms in vitro.Tese encouraging results suggest the potential utility of monolaurin as antibioflm coatings on medical devices to reduce bioflm formation and infections.Despite promising in vitro results, the efectiveness of monolaurin against MRSA bioflms in clinical practice may face challenges related to formulation optimization.Formulating monolaurin into stable and bioavailable preparations suitable for clinical use may pose technical challenges due to its poor solubility in water.

Conclusion
In this study, we evaluated the antibacterial efectiveness and potential synergistic efects of combining monolaurin with β-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) isolates.Te results indicated that monolaurin exhibits signifcant antibacterial activity.Moreover, employing this combination may allow for the reduction of individual antibiotic dosages required and potentially diminish the development of antibiotic resistance.Our study revealed the favorable antibacterial effcacy of monolaurin against clinical strains of MRSA known for their bioflm-producing capabilities.In addition, the study demonstrated an efcient eradication of pre-existing bioflms.Furthermore, a striking correlation was found between elevated monolaurin concentrations and a concomitant decrease in icaD gene expression levels.Henceforth, we propose consistent monitoring of bioflm development in MRSA strains obtained from wound specimens, along with an assessment of their patterns of antimicrobial resistance.Such surveillance could facilitate the establishment of a comprehensive antimicrobial strategy aimed at promptly addressing wound infections.Tis study investigated the impact of varying concentrations of monolaurin (250, 500, 1000, and 2000 μg/mL) on MRSA bioflms.While fndings demonstrated dose-dependent inhibition of bioflm formation, it is imperative to evaluate the practical implications for clinical application.Factors such as monolaurin availability, manufacturing challenges, associated costs, and potential dose-dependent side efects must be considered to align in vitro fndings with in vivo outcomes accurately.Tus, while the study contributes valuable insights into monolaurin's therapeutic potential against MRSA bioflms, further research is essential to assess its feasibility and efectiveness in real-world medical settings.

Figure 1 :
Figure 1: Te optical density (OD) of bioflms formed upon exposure to varying concentrations of monolaurin by MRSA isolates (n � 103).Te experiment was performed in triplicates.Grey color indicates control (OD of MRSA before treatment).Red color indicates OD after treatment with 250 μg/mL of monolaurin.Green color indicates OD after treatment with 500 μg/mL of monolaurin.Violet color indicates OD after treatment with 1000 μg/mL of monolaurin.Blue color indicates OD after treatment with 2000 μg/mL of monolaurin.* Signifcant diference at p value <0.05.

Figure 2 :
Figure 2: Te optical density (OD) of bioflms developed following exposure to diferent concentrations of monolaurin by MRSA isolates (n � 103).Te experiment was performed in triplicates.Grey color indicates control (OD of MRSA before treatment).Red color indicates OD after treatment with 250 μg/mL of monolaurin.Green color indicates OD after treatment with 500 μg/mL of monolaurin.Violet color indicates OD after treatment with 1000 μg/mL of monolaurin.Blue color indicates OD after treatment with 2000 μg/mL of monolaurin.* Signifcant diference at p value <0.05.

Figure 5 :
Figure 5: Efect of sub-MICs of monolaurin on expression of bioflm-related icaD gene among MRSA strains.
3.6.2.Monolaurin Activity against Bioflm Formation of MRSA.As demonstrated in Table3, monolaurin had a remarkable capacity to prevent the bioflm formation of all MRSA strains when compared to control cells cultured in monolaurin-free media.Optical density (OD) values of the treatment, as shown in Figure2, convincingly demonstrated

Table 2 :
Efect of monolaurin on the preformed bioflm of MRSA isolates.
* Correlated with total no of resistant isolates (no � 103).* * Refer to P value which was <0.001 P values <0.05 refer to signifcant diference.OD, optical density.